Design, synthesis, and evaluation of a new fluorescent probe for measuring polymyxin–lipopolysaccharide binding interactions

Abstract

Fluorescence assays employing semisynthetic or commercial dansyl-polymyxin B have been widely employed to assess the affinity of polycations, including polymyxins, for bacterial cells and lipopolysaccharide (LPS). The five primary γ-amines on diaminobutyric acid residues of polymyxin B are potentially derivatized with dansyl-chloride. Mass spectrometric analysis of the commercial product revealed a complex mixture of di- or tetra-dansyl-substituted polymyxin B. We synthesized a mono-substituted fluorescent derivative, dansyl[Lys] 1polymyxin B 3. The affinity of polymyxin for purified gram-negative LPS and whole bacterial cells was investigated. The affinity of dansyl[Lys] 1polymyxin B 3 for LPS was comparable to polymyxin B and colistin, and considerably greater ( K d < 1 μM) than for whole cells ( K d ∼ 6–12 μM). Isothermal titration calorimetric studies demonstrated exothermic enthalpically driven binding between both polymyxin B and dansyl[Lys] 1polymyxin B 3 to LPS, attributed to electrostatic interactions. The hydrophobic dansyl moiety imparted a greater entropic contribution to the dansyl[Lys] 1polymyxin B 3–LPS reaction. Molecular modeling revealed a loss of electrostatic contact within the dansyl[Lys] 1polymyxin B 3–LPS complex due to steric hindrance from the dansyl[Lys] 1 fluorophore; this corresponded with diminished antibacterial activity (MIC ⩾16 μg/mL). Dansyl[Lys] 1polymyxin B 3 may prove useful as a screening tool for drug development.